Patterns of protease production during prostate cancer progression: proteomic evidence for cascades in a transgenic model.

Extracellular proteases are recognized as critical factors in the progression of a number of carcinomas, including prostate cancer. Matrix metalloproteases (MMP) are important in processes of tumor growth, invasion and dissemination, but other classes of proteases, such as serine and cysteine proteases, also contribute. We utilized the TRAMP model for prostate cancer to elucidate proteases involved in prostate cancer progression. General proteomic analysis was performed on normal murine prostate, early TRAMP tumors and advanced TRAMP tumors, as well as normal and involved lymph nodes. Zymography and antigenic analyses revealed increased expression of mainly pro-MMP in early TRAMP tumors but substantial elaboration of activated MMP only in late TRAMP tumors. Progressive increase in cysteine, serine and certain membrane-bound proteases from normal to early to advanced prostate tumors, was also seen. Our results implicate pericellular proteases as initiators of major proteolytic cascades during tumor progression and suggest targets for maximal therapeutic effect.

Vascular endothelial growth factor and basic fibroblast growth factor urine levels as predictors of outcome in hormone-refractory prostate cancer patients: a cancer and leukemia group B study.

Better prognostic markers are needed for hormone-refractory prostate cancer (HRPC) patients. No single biochemical or clinical parameter can reliably predict patient response to therapy or rapidity of disease progression. Peptide factors involved in major cancer growth pathways, such as tumor angiogenesis, are attractive candidates as markers of low- and high-risk HRPC patients. We analyzed prospectively collected urine specimens from 100 of 390 HRPC patients undergoing therapy with the growth factor antagonist suramin as part of CALGB 9480. Levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assessed from day 1 of therapy (D1) and day 29 (D29) urine samples from this subset of 100 randomly selected patients. Growth factor levels were determined by standardized ELISA microtiter plate assays from a commercial (bFGF) or proprietary (VEGF) source. Pretreatment urine VEGF levels were predictive of survival. In univariate analysis, patients whose baseline urine VEGF level was < or =28 pg/ml (the median level) had an average survival of 17 months; those with baseline VEGF >28 pg/ml had a significantly shorter survival of 10 months (P = 0.024). This difference corresponded to a 60% increased risk of dying for the higher urine VEGF patients (hazard ratio, 1.62; P = 0.03) and remained significant in multivariate analysis (hazard ratio, 1.72, P = 0.02). No significant correlations between urine bFGF level or change in bFGF levels and survival were found. These results support the notion that certain peptide growth factor-mediated, mitogenic pathways are important in HRPC and that their levels can predict outcome.

Anti-VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption.

Vascular endothelial growth factor (VEGF) is an important mediator of the intense angiogenesis which is characteristic of glioblastoma. While genetic manipulation of VEGF/VEGF receptor expression has previously been shown to inhibit glioblastoma growth, to date, no study has examined the efficacy of pharmacologic blockade of VEGF activity as a means to inhibit intracranial growth of human glioblastoma. Using intraperitoneal administration of a neutralizing anti-VEGF antibody, we demonstrate that inhibition of VEGF significantly prolongs survival in athymic rats inoculated in the basal ganglia with G55 human glioblastoma cells. Systemic anti-VEGF inhibition causes decreased tumor vascularity as well as a marked increase in tumor cell apoptosis in intracranial tumors. Although intracranial glioblastoma tumors grow more slowly as a consequence of anti-VEGF treatment, the histologic pattern of growth suggests that these tumors adapt to inhibition of angiogenesis by increased infiltration and cooption of the host vasculature.

Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue.

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Thrombopoietin levels in patients with cirrhosis before and after orthotopic liver transplantation.

BACKGROUND: Thrombocytopenia is a common manifestation of cirrhosis. OBJECTIVES: To determine plasma thrombopoietin levels in cirrhotic patients with thrombocytopenia, monitor those levels before and after orthotopic liver transplantation, and compare thrombopoietin messenger RNA (mRNA) levels in liver samples from cirrhotic patients and controls. DESIGN: A cross-sectional study of patients with cirrhosis, including a small subset of patients who had orthotopic liver transplantation. SETTING: University-affiliated hospital. PATIENTS: 44 patients with cirrhosis, including 17 patients who had orthotopic liver transplantation. INTERVENTION: Orthotopic liver transplantation. MEASUREMENTS: Plasma thrombopoietin levels in all patients, platelet counts in all patients, and thrombopoietin mRNA levels in liver samples from nine patients with cirrhosis and eight controls. RESULTS: Thrombopoietin levels were undetectable in 39 of 44 patients with cirrhosis. In 16 of 17 patients, the levels became detectable after liver transplantation. Thrombopoietin mRNA levels were decreased in liver samples from patients with cirrhosis compared with controls (P = 0.0103). CONCLUSIONS: The low thrombopoietin levels in cirrhotic patients with thrombocytopenia and the increased levels after orthotopic liver transplantation suggest that impaired production of thrombopoietin may contribute to thrombocytopenia associated with cirrhosis.

Inhibition of prostate cancer neovascularization and growth by urokinase-plasminogen activator receptor blockade.

Binding of the serine protease urokinase (u-PA) to its receptor on tumor cell surfaces facilitates proteolysis and tumor invasion. We undertook this study to determine whether the role of u-PA in prostate cancer induced angiogenesis and secondary tumor growth by developing a homologous, immunocompetent in vivo model in which the tumors cells secrete an inhibitor of the murine u-PA receptor. A mutant recombinant murine u-PA that retains receptor binding but not proteolytic activity was made by PCR mutagenesis. Mutant u-PA and a reporter gene pRK luciferase were transfected and stably expressed in the highly metastatic rat Dunning MAT-LyLu prostate cancer cell line. Several clones expressing mutant u-PA and luciferase were identified by Western blotting, plasminogen zymography, and reverse transcription-PCR. One of these clones, 5C4, was injected s.c. into Copenhagen rats. Compared to animals injected with clones expressing pRK luciferase alone, tumors in animals injected with 5C4 cells were significantly smaller. Moreover, there were fewer lung micrometastases in the 5C4 animals. Primary tumor angiogenesis was measured by microvessel quantification of tissue stained with antibodies against von Willebrand factor. Mean microvessel density in 5C4 tumors was 4.3-fold lower than that in animals with tumors derived from the control tumor cell line (P < 0.0001). Significant inhibition of tumor growth was also observed for two additional MAT-LyLu cell lines expressing mutant u-PA. These findings suggest that cell surface u-PA contributes to prostate cancer growth by enhancing angiogenesis.

Decreased prostate cancer cell migration by inhibition of the insulin-like growth factor II/Mannose-6-Phosphate receptor.

The 270-kDa insulin-like growth factor II (IGF-II)/cation-independent mannose-6-phosphate receptor (MPR) is a multifunctional receptor protein. Endocytoses and intracellular transport of soluble enzymes bearing mannose6-phosphate (M-6-P) residues to lysosomes is mediated by the IGF-II/MPR. We examined human prostate cancer cells for IGF-II/MPR expression to determine whether this receptor mediates cell migration. PC3 human prostate cancer cells were studied for intracellular IGF-II/MPR by immunoblotting. PC3 cell surface IGF-II/MPR expression was assessed by flow cytometric analysis. Cell motility was quantitated by a scratch migration assay, and IGF-II/MPR blockade was achieved using M-6-P or affinity-purified rabbit anti-bovine cation-independent IGF-II/MPR immunoglobulin. IGF-II/MPR is expressed in the cytoplasm and on the surface of PC3 prostate cancer cells. The mean number of PC3 cells migrating per high powered field in medium containing polyclonal anti-IGF-II/MPR immunoglobulin or M-6-P decreased significantly (5 ± 4 cells and 34 ± 5 cells, respectively) compared with control medium containing mouse immunoglobulin G (70 ± 12 cells) or mannose-1-phosphate (67 ± 7 cells). This decreased PC3 cell migration following cell surface IGF-II/MPR blockade suggests that the IGF-II/MPR may play an important role in prostate cancer cell motility.

Circulating thrombopoietin concentrations in thrombocytopenic patients, including cancer patients following chemotherapy, with or without peripheral blood progenitor cell transplantation.

Thrombopoietin, the ligand for the c-mpl receptor, promotes proliferation and maturation of megakaryocytes. An ELISA using a chimaeric receptor, mpl-IgG, for capture, and rabbit antibody to thrombopoietin for detection was developed for the quantitation of thrombopoietin in human serum or plasma. This ELISA preferentially detects full-length thrombopoietin compared to the bioactive N-terminal half of the molecule which has homology to erythropoietin. Thrombopoietin was not detected (< 0.16 ng/ml) in 88/89 healthy individuals. However, elevated thrombopoietin concentrations of up to 3 ng/ml were detected in 59/63 thrombocytopenic patients, including cancer patients following chemotherapy. In cancer patients receiving chemotherapy with (n = 12) or without (n = 6) peripheral blood progenitor cell transplantation, thrombopoietin concentrations varied inversely with platelet counts throughout the treatment period. In general, patients who received myeloablative chemotherapy on days -7 to -2 and peripheral blood progenitor cell transplantation on day 0 had high thrombopoietin levels (0.6-2.9 ng/ml) around day 5. Low platelet counts (< 20 x 10(9)/l) occurred between days 4 and 9. Patients who received high-dose chemotherapy on day 1 (equivalent to day -7 for transplantation patients) to day 6 without transplantation had high thrombopoietin concentrations (1.4-2.3 ng/ml) around day 13 and low platelet counts occurred between days 7 and 17.

Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice.

Urokinase plasminogen activator (uPA) and its receptor are key components of a cell surface proteolytic cascade used by tumor cells and capillary endothelial cells for basement membrane invasion, a process required for metastasis and angiogenesis. We have cloned, expressed, and purified the epidermal growth factor-like domain of murine uPA alone and fused it to the Fc portion of human IgG as high-affinity murine urokinase receptor antagonists. These molecules are potent inhibitors of murine urokinase binding to its receptor and inhibit angiogenesis in an in vitro model of capillary tube formation in fibrin gels. In vivo, basic fibroblast growth factor-induced neovascularization and B16 melanoma growth in syngeneic mice are also substantially suppressed by these molecules. Coupled with previous studies showing inhibition of metastasis, these findings suggest that urokinase receptor antagonists may be useful therapeutically as inhibitors of tumor progression.

Vascular endothelial growth factor promotes tumor dissemination by a mechanism distinct from its effect on primary tumor growth.

Tumor growth is dependent on new blood vessel formation. Inhibition of vascular endothelial growth factor (VEGF), an endothelial cell mitogen and angiogenic factor secreted by a variety of tumors and tumor cell lines, is sufficient to inhibit primary tumor growth. In the present study, we examined the effect of inhibiting VEGF on tumor cell micrometastasis. A transfectant of A431 (a human epidermoid carcinoma cell line) expressing chloramphenicol acetyltransferase (CAT) was injected s.c. into severe combined immunodeficiency (scid) mice, which were then sacrificed after 6 weeks. The presence of A431 metastases at distant sites was demonstrated by detection of CAT activity in whole-organ lysates. Treatment of animals with VEGF-neutralizing antibodies not only inhibited primary tumor growth but also suppressed metastases, as determined by CAT activity in organ lysates. In experiments to determine the mechanism by which anti-VEGF antibody inhibited metastasis, control animals were sacrificed when their tumors had reached the same size as tumors in VEGF antibody-treated animals. Metastases were uniformly present in these control animals. These findings show that inhibition of VEGF alone is sufficient to prevent tumor growth and dissemination in vivo. The inhibitory effect on metastases appears to be distinct from that on primary tumor growth.

Thrombin increases expression of urokinase receptor by activation of the thrombin receptor.

PURPOSE: To investigate the effect of thrombin on the urokinase plasminogen activator receptor (u-PAR) in retinal pigment epithelial (RPE) cells. METHODS: The authors analyzed u-PAR mRNA by Northern blot hybridization. Retinal pigment epithelial cell surface u-PAR was assayed by measuring the amount of functional urokinase plasminogen activator (u-PA) bound to cells at saturation. Retinal pigment epithelial cells were derived from fetal retinal tissue and established in primary cell culture. RESULTS: Thrombin increased u-PAR mRNA 4-fold in RPE cells examined by Northern blot hybridization, whereas the amount of thrombin receptor mRNA was unchanged. Thrombin stimulated u-PA binding to RPE cells 2.5- to 5-fold in a time- and dose-dependent manner. Hirudin, a thrombin antagonist, completely blocked the effects of thrombin on u-PAR expression in RPE cells. Phosphatidylinositol phospholipase C treatment of RPE cells resulted in the abolition of thrombin-induced u-PA binding. Recombinant soluble u-PAR competitively inhibited two-chain u-PA binding to the surface of thrombin-treated RPE cells. A thrombin receptor agonist peptide (SFLLRNPNDKYEPF) also induced a 2.5-fold increase in binding of u-PA to the surface of RPE cells. CONCLUSION: Thrombin increases u-PAR expression by RPE cells by a mechanism involving activation of the seven transmembrane thrombin receptor.

Endocytosis of fibrinogen into hamster megakaryocyte alpha granules is dependent on a dimeric gamma A configuration.

Two species of fibrinogen that differ only in the structure of their gamma chains, gamma A and gamma', are present in normal plasma. Fibrinogen stored in platelet alpha granules does not contain gamma' chains. Because platelet fibrinogen was recently shown to be derived exclusively by receptor-mediated endocytosis from plasma and not by endogenous megakaryocyte synthesis, we postulated that the gamma' fibrinogen present in plasma is not endocytosed by megakaryocytes and platelets. We tested this hypothesis by studying endocytosis of peak 1 (containing two gamma A chains) and peak 2 (containing one gamma A and one gamma' chain) fractions of human fibrinogen obtained from diethyl aminoethyl (DEAE) cellulose chromatography in an in vivo hamster model. When 10 mg of biotinylated, unfractionated, or peak 1 fibrinogen was injected intravenously, each protein was endocytosed into megakaryocytes and platelets within 24 hours. In contrast, equivalent doses of biotinylated peak 2 fibrinogen and bovine serum albumin were barely detectable within megakaryocytes and platelets. We conclude that gamma' fibrinogen is not endocytosed and incorporated into megakaryocytes and platelet alpha granules. Furthermore, a dimeric gamma A-chain configuration is required for receptor-mediated endocytosis of fibrinogen into these organelles.

Immunohistochemical localization of the plasminogen activator inhibitor-1 in breast cancer.

We used an immunohistochemical assay with an antigen-retrieval technique to study plasminogen activator inhibitor type-1 (PAI-1) expression in paraffin-embedded breast tissue samples at different stages of malignant transformation. We detected PAI-1 in 15/20 invasive tumors. In several cases staining was localized to the stromal component. PAI-1-positive fibroblasts could be seen surrounding tumor nodules or at tumor margins. In addition, tumor-infiltrating macrophages (13 cases) and endothelial cells (5 cases) were positive. In 11 specimens PAI-1-positive cancer cells were also detected. In 2 strongly positive cases secreted PAI-1 was visible in the extracellular matrix surrounding the cells. Six of 9 samples of carcinoma in situ (DCIS) were weakly positive. No staining of endothelial cells was visible in DCIS. Only a few positive adenomatous epithelial cells could be seen in 3 of 7 papillomas. All biopsies of normal breast tissue were negative, with the exception of one sample, obtained from a patient with a previous segmental mastectomy for DCIS. PAI-1 production by invasive breast cancers could reflect a general upregulation of the plasminogen activation system in proliferating cancer cells, as suggested by the finding that normal mammary epithelium cultures expressed PAI-1 in all cases examined. In addition, production of PAI-1 by the tumor stroma could protect the tumor itself from excessive proteolysis.

Co-expression of urokinase, urokinase receptor and PAI-1 is necessary for optimum invasiveness of cultured lung cancer cells.

We investigated the importance of the urokinase (uPA)-plasmin system in fostering invasion of human lung cancer cells through artificial basement membranes composed of Matrigel. Eight cell lines (including 1 small cell and 7 non-small cell lines) were examined. One cell line did not express any components of the urokinase system. Four cell lines had substantial levels of endogenous uPA detectable on their surfaces. Three of these cell lines co-expressed the plasminogen activator inhibitor PAI-1 in addition to uPA. Assays for invasiveness revealed 4 cell lines capable of traversing a Matrigel barrier, including the 3 which co-expressed uPA, PAI-1 and uPA receptor. Surprisingly, the cell line expressing only uPA and uPA receptor displayed no invasive capacity despite levels of secreted uPA more than 20-fold higher than the other cell lines studied. Based on these observations, we hypothesized that both uPA and PAI-1 might be important for invasion by lung tumor cells, at least in vitro. We therefore tested polyclonal antibodies which inhibit uPA and PAI-1 activity for their effects on the highly invasive H292 cell line. After 3 days, invasive capacity was inhibited by antibodies to both uPA and PAI-1 in a dose-dependent manner. The plasmin inhibitor aprotinin reduced H292 cell invasion by 70%. Taken together, our data demonstrate that in cultured human lung cancer cells the uPA-plasmin system is important in promoting invasion into basement membranes and suggest that a critical balance between uPA and PAI-1 is necessary for optimal invasiveness. Our data are consistent with results from recent clinical studies showing that PAI-1 expression in tumor tissue is an adverse prognostic feature.

Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals.

OBJECTIVE: To characterize a Kaposi's sarcoma (KS) cell line established from a tumor biopsy from the oral mucosa of an iatrogenically immunosuppressed HIV-negative man. METHODS: Cells were placed in culture and evaluated by a variety of biologic, serologic, karyotypic, and immunologic procedures. Electron microscopic examination was performed. The ability to produce tumors in nude mice was evaluated, and the nature of the cells within the tumor determined. Assays for urokinase plasminogen activator type (uPA), plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR) were conducted. RESULTS: The SLK cell line has an endothelial cell morphology with very little anaplasia. The karyotype indicates diploid phenotype of human origin. Immunohistochemical and electron microscopic examinations confirmed the endothelial nature of this cell line. No viruses were detected. The tumors induced in nude mice showed hypervascularization, with characteristics of KS. The cell line produces uPA and PAI-1, and also expresses uPAR. CONCLUSIONS: The SLK cell line is of endothelial cell origin and the first human cell line to induce KS-like tumors in recipient animals. The expression of urokinase and its receptor suggests a paracrine and autocrine interaction that may be important for the growth of the tumor. The SLK line should be valuable for studies of KS pathogenesis and therapeutic approaches to this malignancy.

The urokinase receptor is expressed in invasive breast cancer but not in normal breast tissue.

We have studied expression of the urokinase receptor (u-PAR) in paraffin-embedded breast tissues at various stages of malignant progression. Forty-nine of 59 invasive cancers studied showed varying degrees of reactivity with our polyclonal antibody. The staining pattern was variable from case to case, although strong surface staining of tumor-associated macrophages was evident in most of these sections. In several cases, blood vessels in selected tumor areas were stained, as confirmed by treatment of adjacent sections with an anti-factor VIII antibody. These could represent regions of recent angiogenesis. Staining of tumor cells was observed in 21 of 59 cases and was extensive in 5 cases but confined to a small percentage of cells in the remaining 16 samples. In contrast with the cancer sections, all normal breast tissue (12 cases) was negative, as well as all fibroadenomas (4 cases), papillomas (5 cases), and hyperplasia with atypia (2 cases) studied. Seven carcinomas in situ examined were also negative for u-PAR, with the exception of few macrophages in two cases, suggesting that u-PAR expression may be associated with invasive tumor. The presence of u-PAR in human breast cancer and its absence from nonmalignant breast tissue supports the idea that plasminogen activation plays an important role in the process of cancer invasion. Expression of u-PAR on macrophages, endothelial cells, and cancer cells suggests the existence of complex paracrine interactions between tumor cells and stroma.

Endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is mediated by alpha IIb beta 3 (glycoprotein IIb-IIIa)

Recently, we showed that platelet alpha-granule fibrinogen is derived entirely from endocytic uptake and not from megakaryocyte synthesis, as previously thought. In this present report, we identify the receptor that mediates endocytosis. We have found that barbourin, a unique disintegrin that is a specific antagonist of alpha IIb beta 3, inhibits the endocytic uptake of fibrinogen into alpha-granules. Continuous intravenous infusion of barbourin (200 micrograms/h) into guinea pigs blocked collagen-induced platelet aggregation as well as endocytosis of biotinylated fibrinogen into megakaryocytes; however, endocytosis of biotinylated albumin by megakaryocytes was not affected. Thus, we have shown that endocytosis of fibrinogen into megakaryocyte and platelet alpha-granules is receptor-mediated, and that alpha IIb beta 3 is the primary receptor.

Prevention of metastasis by inhibition of the urokinase receptor.

The plasminogen activator urokinase (u-PA) mediates proteolysis by a variety of human tumor cells. Competitive displacement of u-PA from cellular binding sites results in decreased proteolysis in vitro, suggesting that the cell surface is the preferred site for u-PA-mediated protein degradation. We studied the effect of u-PA receptor blockade on the metastatic capacity of human PC3 prostate carcinoma cells, using transfectants which expressed chloramphenicol acetyl-transferase (CAT). Eight weeks after subcutaneous inoculation of these cells into nude mice, CAT activity was detected in regional lymph nodes, femurs, lungs, and brain, thereby mimicking the organ tropism observed for naturally occurring metastases of prostate cancer. In a second transfection, CAT-expressing PC3 cells received cDNA encoding a mutant u-PA (Ser356-->Ala) which lacks enzymatic activity but which retains full receptor binding affinity. Three mutant u-PA expressors, each with < 5% of wild-type cell-associated u-PA activity, were compared in vivo with independently derived controls. Primary tumor growth was similar in each group of animals and all tumors expressed comparable CAT activity. In contrast, metastasis (as assessed by CAT activity) was markedly inhibited when cell surface u-PA activity was blocked. Levels of CAT activity were reduced by a factor of > 300 in regional lymph nodes, 40-100 in brain tissue, and 10-20 in lung tissue. Metastatic capacity was inhibited similarly when animals were given intermittent intraperitoneal injections of a u-PA/IgG fusion protein capable of displacing u-PA activity from the tumor cell surface. Our results indicate that cell surface u-PA activity is essential to the metastatic process. In addition, the assay system employed in these experiments may be generally useful in testing other therapeutic modalities to limit the spread of primary tumors.